The present invention relates to a composition for use in screening toxicological effects of test compounds or therapeutic treatments and methods employing such compositions.
Toxicity testing is a necessary and time-consuming part of the pharmaceutical drug development pipeline. A more rapid screen to detect toxicity of lead drug candidates may be the use of gene expression microarrays. For example, microarrays consisting of full length genes or gene fragments on a substrate may be formed. These arrays can then be tested with samples treated with the drug candidates to elucidate the gene expression pattern associated with treatment with the drug candidate. This gene pattern can be compared with gene expression patterns of compounds associated with known toxicological responses.
Benzo(a)pyrene (BP) is a known rodent and likely human carcinogen and is the prototype of a class of compounds, the polycyclic aromatic hydrocarbons (PAHs). It is metabolized by several forms of cytochrome P450 and associated enzymes to both activated and detoxified metabolites (Degawa et al. (1994) Cancer Res. 54: 4915-4919). The ultimate metabolites are the bay-region diol epoxide, benzo(a)pyrene-7,8-diol-9,10-epoxide (BPDE) and the K-region diol epoxide, 9-hydroxy benzo(a)pyrene-4,5-oxide, which have been shown to form DNA adducts. BPDE-DNA adducts have been shown to persist in rat liver up to 56 days post dose with the treatment regimen of 10 mg/kg b.w. 3 times per week for 2 weeks (Qu and Stacey (1996) Carcinogenesis 17: 53-59). It has recently been shown that the BPDE-DNA adduct preferentially binds to methylated CpG sites in the p53 gene at sites where mutations are known to occur (Chen et al. (1998) Cancer Res. 58:2070-2075). Mutations in this tumor suppressor gene have been discovered in over 50% of human cancers (Greenblatt et al. (1994) Cancer Res. 54: 4855-4878).
Acetaminophen (APAP) is a well-recognized and widely-used analgesic. It is metabolized by specific cytochrome P450 isozymes with the majority of the drug undergoing detoxification by glucuronic acid, sulfate and glutathione conjugation pathways (Chen et al. (1998) Chem. Res. Toxicol. 11: 295-301). However, at large nontherapeutic doses, APAP can cause hepatic and renal failure by being metabolized to an active intermediate, N-acetyl-p-benzoquinone imine (NAPQI). NAPQI then binds to sulfhydryl groups of proteins causing their inactivation and leading to subsequent cell death (Kroger et al. (1997) Gen. Pharmacol. 28: 257-263).
Clofibrate (CLO) is an antilipidemic drug which lowers elevated levels of serum triglycerides. In rodents, chronic treatment produces hepatomegaly, an increase in hepatic peroxisomes and has been shown to be a hepatocarcinogen but not a mutagen (Lock et al. (1989) Ann. Rev. Pharmacol. Toxicol. 29:145). CLO has been shown to induce cytochrome P450 4A and reduce the levels of P450 4F (Kawashima et al. (1997) Arch. Biochem. Biophys. 347:148-154). It is also involved in transcription of b-oxidation genes as well as induction of peroxisome proliferator activated receptors (PPARs) (Kawashima supra).
The present invention provides compositions and methods for screening, preferably in a microarray format, of compounds and therapeutic treatments for toxicological effects.
In one aspect, the invention provides a method for screening a compound for a toxicological effect. The method comprises (i) selecting a plurality of polynucleotide targets, wherein said polynucleotide targets have first gene expression levels altered in tissues treated with known toxicological agents when compared with untreated tissues, (ii) treating a sample with the compound to induce second gene expression levels of a plurality of polynucleotide probes, and (iii) comparing the first and second gene expression levels to identify those compounds that induce expression levels of the polynucleotide probes that are similar to those of the polynucleotide targets and said similarity of expression levels correlates with a toxicological effect of the compound.
Preferably, the comparing comprises (i) contacting said polynucleotide targets with the polynucleotide probes under conditions effective to form hybridization complexes between said polynucleotide targets and said polynucleotide probes, and (ii) detecting the presence or absence of said hybridization complexes. In this context, similarity may mean that at least 1, preferably at least 5, more preferably 10, of the upregulated polynucleotide targets form hybridization complexes with the polynucleotide probes at least once during a time course to a greater extent than would the probes of a sample not treated with the test compound. Similarity may also mean that at least 1, preferably at least 3, of the downregulated polynucleotide target sequences form hybridization complexes with the polynucleotide probes at least once during a time course to a lesser extent than would the probes of a sample not treated with the test compound.
Preferred tissues are selected from the group consisting of liver, kidney, brain, spleen, pancreas and lung. Preferred toxicological agents are selected from the group consisting of benzo(a)pyrene, methylcholanthrene, benz(a)anthracene, 7,12-dimethylbenz(a)anthracene and their corresponding toxic metabolites. The polynucleotide targets comprise genes that are upregulated-or-down regulated at least 2 fold, preferably at least 3 fold, in tissues treated with known toxicological agents when compared with untreated tissues. Preferred polynucleotide targets are selected from the group consisting of SEQ ID NOs: 1-47, or fragments thereof, some of whose expression is upregulated and others of whose expression is downregulated. Even more preferable are SEQ ID NOs: 2, 8, 10, 13, 19, 26, 31, 33, 35, 37, 39, and 42 which are upregulated and SEQ ID Nos: 11, 25, 27, 28, and 45 which are downregulated. In one embodiment, the polynucleotide targets are hybridizable array elements of a microarray.
Alternatively, the invention provides methods for screening a therapeutic treatment for a toxicological effect or for screening a sample for a toxicological response to a compound or therapeutic treatment.
In another aspect, the invention provides methods for preventing a toxicological response by administering complementary nucleotide sequences against one or more selected upregulated polynucleotide target or a ribozyme that specifically cleaves such sequences. Alternatively, a toxicological response may be prevented by administering sense nucleotide sequences for one or more selected down regulated polynculeotide targets.
A portion of the disclosure of this patent document contains material which is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as it appears in the Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever.
The Sequence Listing contains the sequences of exemplary polynucleotide targets of the invention, SEQ ID NOs: 1-47.
Definitions
The term xe2x80x9cmicroarrayxe2x80x9d refers to an ordered arrangement of hybridizable array elements. The array elements are arranged so that there are preferably at least one or more different array elements, more preferably at least 10 array elements, and most preferably at least 100 array elements, and even more preferably 10,000, on a 1 cm2 substrate surface. Furthermore, the hybridization signal from each of the array elements is individually distinguishable. In a preferred embodiment, the array elements comprise polynucleotide sequences.
A xe2x80x9cpolynucleotidexe2x80x9d refers to a chain of nucleotides. Preferably, the chain has from about 5 to 10,000 nucleotides, more preferably from about 50 to 3,500 nucleotides. The term xe2x80x9cpolynucleotide targetxe2x80x9d refers to a polynucleotide sequence capable of hybridizing with a xe2x80x9cpolynucleotide probexe2x80x9d to form a polynucleotide target/probe complex under hybridization conditions.
In some instances, the sequences will be complementary (no mismatches) when aligned. In other instances, there may be a substantial mismatch, up to 10%.
A xe2x80x9cpluralityxe2x80x9d refers preferably to a group of one or more members, preferably to a group of at least about 10, and more preferably to a group of at least about 100 members, even more preferably a group of 10,000 members.
The term xe2x80x9cgenexe2x80x9d or xe2x80x9cgenesxe2x80x9d refers to the partial or complete coding sequence of a gene. The term also refers to 5xe2x80x2 or 3xe2x80x2 untranslated regions. The gene may be in a sense or antisense configuration.
xe2x80x9cToxicological agent or compoundxe2x80x9d is any compound that elicits an unfavorable response in an individual or animal, such as DNA damage, organ damage, cell damage or cell death.
A xe2x80x9cfragmentxe2x80x9d refers to a sequence which is a portion of a polynucleotide target sequence. Exemplary fragments are sequences comprising nucleotides 1-20 of SEQ ID Nos: 1-47.
The Invention
The present invention provides a composition and method for screening test compounds or therapeutic treatments for toxicological effects or for characterizing the toxicological responses of a sample to a test compound or a therapeutic treatment. In particular, the present invention provides a composition comprising a plurality of polynucleotide sequences derived from normal rat liver cDNA libraries, normalized rat liver cDNA libraries and prehybridized rat liver cDNA libraries and rat kidney libraries. The polynucleotide sequences have been further selected for exhibiting up-or-down regulated gene expression in rat liver samples when the rat liver samples have been exposed to a known toxin, in particular a hepatotoxin and more particularly a polycyclic aromatic hydrocarbon (PAH). PAHs include compounds such as benzo(a)pyrene, 3-methylcholanthrene, benz(a)anthracene, 7,12-dimethylbenz(a)anthracene, their corresponding metabolites, and the like. In a preferred embodiment the toxin is benzo(a)pyrene, or one of its toxic metabolites. The extent of upregulation or downregulation is at least 2 fold, more preferably at least 3 fold.
Exemplary polynucleotide sequences (targets) include SEQ ID NOs: 1-47 provided in the Sequence Listing or fragments thereof. These and other polynucleotide sequences can be immobilized on a substrate and used as hybridizable array elements in a microarray format. The microarray may be used to characterize gene expression patterns associated with novel compounds to elucidate any toxicological effects or to monitor the effects of therapeutic treatments where toxicological effects may be expected.
When the polynucleotide targets are employed as hybridizable array elements in a microarray, the array elements are organized in an ordered fashion so that each element is present at a specified location on the substrate. Because the array elements are at specified locations on the substrate, the hybridization patterns and intensities (which together create a unique expression profile) can be interpreted in terms of expression levels of particular genes and can be correlated with a toxicological effect associated with a compound or a therapeutic treatment.
Furthermore, the present invention provides methods for screening compounds and/or therapeutic treatments for potential toxicological effects and for screening a sample""s toxicological response to a particular test compound. Briefly, these methods entail treating a sample with the compound to be tested to elicit a change in gene expression patterns comprising the expression of a plurality of polynucleotide probes. Polynucleotide targets are selected by identifying those genes in rat liver or kidney that are up-or-downregulated at least 2 fold, more preferably at least 3 fold, when treated with a known toxic compound. Then, the polynucleotide targets and probes are combined under conditions effective to form hybridization complexes which may be detected by methods well known in the art. Detection of higher or lower levels of such hybridization complexes compared with hybridization complexes derived from samples treated with a compound that is known not to induce a toxicological effect correlates with a toxicological effect of a test compound or a toxicological response to a therapeutic treatment.
Sequences are identified that reflect all or most of the genes that are expressed in rat liver or kidney. Sequences may be identified by isolating clones derived from several types of rat cDNA libraries, including normal rat cDNA libraries, normalized rat cDNA libraries and prehybridized rat cDNA libraries. Clone inserts derived from these clones may be partially sequenced to generate expressed sequence tags (ESTs).
In one embodiment, two collections of ESTs are identified and sequenced. A first collection of ESTs (the originator sequences) are derived from rat liver and kidney and are derived from the cDNA libraries presented in the Examples. A second collection includes ESTs derived from other rat cDNA libraries available in the ZOOSEQ database (Incyte Genomics, Palo Alto, Calif.).
The two collections of ESTs are screened electronically to form master clusters of ESTs and then further analyzed as described below. Master clusters are formed by identifying overlapping EST sequences and assembling these ESTs. A nucleic acid fragment assembly tool, such as the Phrap tool (WashU-Merck) and the GELVIEW Fragment Assembly system (GCG), can be used for this purpose. The minimum number of clones necessary to constitute a cluster is two.
In another embodiment, a collection of human genes implicated in toxicology are used as the originator sequences and the collection of ESTs derived from the 55 rat cDNA libraries are again used as the additional sequences. Master clusters are formed around specific originator sequences, including the ESTs identified in rat liver and kidney or GenBank sequences which code for polypeptides implicated in toxicology in humans. After the sequences have been clustered, the most 5xe2x80x2 clone is selected. After assembling the sequences, a representative clone is nominated from each master cluster. The most 5xe2x80x2 clone is usually preferred because it is the one most likely to contain the complete gene. The nomination process is described in greater detail in xe2x80x9cRelational Database and System for Storing Information Relating to Biomolecular Sequences and Reagents, Ser. No. 09/034,807, filed Mar. 4, 1998, herein incorporated in its entirety by reference.
Then, samples are treated, preferably at subchronic doses, with one or more known toxicological agents over a defined time course. Preferred toxicological agents are hepatotoxins, nephrotoxins, cardiotoxins and the like. Preferably, the agents are hepatotoxins, in particular members of the polycyclic aromatic hydrocarbon class. To this class belong benzo(a)pyrene, methylcholanthrene, benz(a)anthracene, 7,12-dimethylbenz(a)anthracene, their corresponding toxic metabolites, and the like. Alternatively, agents such as clofibrate and acetaminophen may be investigated.
The gene expression patterns derived from such treated biological samples can be compared with the gene expression patterns derived from untreated biological samples to identify genes whose expression is either upregulated or downregulated due to the presence of the toxins. These sequences may then be employed as array elements in a microarray alone or in combination with other array element sequences. Such a microarray is particularly useful to detect and characterize gene expression patterns associated with known toxicological agents. Such gene expression patterns can then be used for comparison to identify other compounds or therapeutic treatments which also elicit a toxicological response.
The selected polynucleotide sequences can be manipulated further to optimize the performance of the polynucleotide sequences as hybridization sequences. Some sequences may not hybridize effectively under hybridization conditions due to secondary structure. To optimize probe hybridization, the probe sequences are examined using a computer algorithm to identify portions of genes without potential secondary structure. Such computer algorithms are well known in the art, such as OLIGO 4.06 Primer Analysis Software (National Biosciences) or LASERGENE software (DNASTAR). These programs can search nucleotide sequences to identify stem loop structures and tandem repeats and to analyze G+C content of the sequence (those sequences with a G+C content greater than 60% are excluded). Alternatively, the sequences can be optimized by trial and error. Experiments can be performed to determine whether sequences and complementary target polynucleotides hybridize optimally under experimental conditions.
The polynucleotide sequences can be DNA or RNA, or any RNA-like or DNA-like material, such as mRNAs, cDNAs, genomic DNA, peptide nucleic acids, branched DNAs and the like. The polynucleotide sequences can be in sense or antisense orientations.
In one embodiment, the polynucleotide sequences are cDNAs. The size of the DNA sequence of interest may vary, and is preferably from 50 to 10,000 nucleotides, more preferably from 150 to 3,500 nucleotides. In a second embodiment, the polynucleotide sequences are vector DNAs. In this case the size of the DNA sequence of interest, i.e., the insert sequence, may vary from about 50 to 10,000 nucleotides, more preferably from about 150 to 3,500 nucleotides.
The polynucleotide sequences can be prepared by a variety of synthetic or enzymatic schemes which are well known in the art. (Caruthers et al. (1980) Nucl. Acids Res. Symp. Ser. 215-233). Nucleotide analogues can be incorporated into the polynucleotide sequences by methods well known in the art. The only requirement is that the incorporated nucleotide analogues must serve to base pair with polynucleotide probe sequences. For example, certain guanine nucleotides can be substituted with hypoxanthine which base pairs with cytosine residues. However, these base pairs are less stable than those between guanine and cytosine. Alternatively, adenine nucleotides can be substituted with 2,6-diaminopurine which can form stronger base pairs than those between adenine and thymidine. Additionally, the polynucleotide sequences can include nucleotides that have been derivatized chemically or enzymatically. Typical modifications include derivatization with acyl, alkyl, aryl or amino groups.
The polynucleotide sequences can be immobilized on a substrate via chemical bonding procedures. Furthermore, the sequences do not have to be directly bound to the substrate, but rather can be bound to the substrate through a linker group. The linker groups are typically about 6 to 50 atoms long to provide exposure to the attached polynucleotide probe. Preferred linker groups include ethylene glycol oligomers, diamines, diacids and the like. Reactive groups on the substrate surface react with one of the terminal portions of the linker to bind the linker to the substrate. The other terminal portion of the linker is then functionalized for binding the polynucleotide probe. Preferred substrates are any suitable rigid or semirigid support, including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which the polynucleotide sequences are bound. Preferably, the substrates are optically transparent.
The samples can be any sample comprising polynucleotide probes and obtained from any bodily fluid (blood, urine, saliva, phlegm, gastric juices, etc.), cultured cells, biopsies, or other tissue preparations. The samples can be derived from humans or animal models.
DNA or RNA can be isolated from the sample according to any of a number of methods well known to those of skill in the art. For example, methods of purification of nucleic acids are described in Laboratory Technicues in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes. Part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed. Elsevier (1993). In one preferred embodiment, total RNA is isolated using the TRIZOL total RNA isolation reagent (Life Technologies, Inc.) and mRNA is isolated using oligo d(T) column chromatography or glass beads. When polynucleotide probes are amplified it is desirable to amplify the nucleic acid sample and maintain the relative abundances of the original sample, including low abundance transcripts. RNA can be amplified in vitro, in situ or in vivo (See Eberwine U.S. Pat. No. 5,514,545).
It is also advantageous to include quantitation controls within the sample to assure that amplification and labeling procedures do not change the true distribution of polynucleotide probes in a sample. For this purpose, a sample is spiked with a known amount of a control polynucleotide and the composition of polynucleotide sequences includes reference polynucleotide sequences which specifically hybridize with the control target polynucleotides. After hybridization and processing, the hybridization signals obtained should reflect accurately the amounts of control polynucleotide added to the sample.
Prior to hybridization, it may be desirable to fragment the polynucleotide probes. Fragmentation improves hybridization by minimizing secondary structure and cross-hybridization to other polynucleotide probes in the sample or noncomplementary polynucleotide sequences. Fragmentation can be performed by mechanical or chemical means.
The polynucleotide probes may be labeled with one or more labeling moieties to allow for detection of hybridized probe/target polynucleotide complexes. The labeling moieties can include compositions that can be detected by spectroscopic, photochemical, biochemical, bioelectronic, immunochemical, electrical, optical or chemical means. The labeling moieties include radioisotopes, such as 32p, 33p or 35S, chemiluminescent compounds, labeled binding proteins, heavy metal atoms, spectroscopic markers, such as fluorescent markers and dyes, magnetic labels, linked enzymes, mass spectrometry tags, spin labels, electron transfer donors and acceptors, and the like. Preferred fluorescent markers include C3 and C5 (Amersham).
Hybridization causes a polynucleotide probe and a complementary target polynucleotide to form a stable duplex through base pairing. Hybridization methods are well known to those skilled in the art (See, for example, Laboratory Techniques in Biochemistry and Molecular Biology, Vol. 24: Hybridization With Nucleic Acid Probes, P. Tijssen, ed. Elsevier, N.Y. (1993)). Conditions can be selected for hybridization where only fully complementary target and polynucleotide probe hybridize, i.e., each base pair must interact with its complementary base pair. Alternatively, conditions can be selected where target and polynucleotide sequences have mismatches but are still able to hybridize. Suitable conditions can be defined by salt concentration, temperature, and other chemicals and conditions well known in the art. Varying additional parameters, such as hybridization time, the concentration of detergent (sodium dodecyl sulfate, SDS) or solvent (formamide), and the inclusion or exclusion of carrier DNA, are well known to those skilled in the art. Additional variations on these conditions will be readily apparent to those skilled in the art (Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) Methods Enzymol. 152:507-511; Ausubel, F. M. et al. (1997) Short Protocols in Molecular Biology, John Wiley and Sons, New York, N.Y.; and Sambrook, J. et al. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).
More particularly, hybridization can be performed with buffers, such as 5xc3x97SCC/0.1% SDS at 60xc2x0 C. for about 6 hours. Subsequent washes are performed at higher stringency with buffers, such as 1xc3x97SCC/0.1% SDS at 45xc2x0 C., then 0.1xc3x97SCC at to retain hybridization of only those target/probe complexes that contain exactly complementary sequences. Alternatively, salt concentration may be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Stringent temperature conditions will ordinarily include temperatures of at least about 22xc2x0 C., more preferably of at least about 37xc2x0 C., and most preferably of at least about 42xc2x0 C.
Hybridization specificity can be evaluated by comparing the hybridization of specificity-control polynucleotide sequences to specificity-control polynucleotide probes that are added to a sample in a known amount. The specificity-control target polynucleotides may have one or more sequence mismatches compared with the corresponding polynucleotide sequences. In this manner, whether only complementary target polynucleotides are hybridizing to the polynucleotide sequences or whether mismatched hybrid duplexes are forming is determined.
Hybridization reactions can be performed in absolute or differential hybridization formats. In the absolute hybridization format, polynucleotide probes from one sample are hybridized to the sequences in a microarray format and signals detected after hybridization complex formation correlate to polynucleotide probe levels in a sample. In the differential hybridization format, the differential expression of a set of genes in two biological samples is analyzed. For differential hybridization, polynucleotide probes from both biological samples are prepared and labeled with different labeling moieties. A mixture of the two labeled polynucleotide probes is added to a microarray. The microarray is then examined under conditions in which the emissions from the two different labels are individually detectable.
Sequences in the microarray that are hybridized to substantially equal numbers of polynucleotide probes derived from both biological samples give a distinct combined fluorescence (Shalon et al. PCT publication WO95/35505). In a preferred embodiment, the labels are fluorescent labels with distinguishable emission spectra, such as C3 and C5 fluorophores.
After hybridization, the microarray is washed to remove nonhybridized nucleic acids and complex formation between the hybridizable array elements and the polynucleotide probes is detected. Methods for detecting complex formation are well known to those skilled in the art. In a preferred embodiment, the polynucleotide probes are labeled with a fluorescent label and measurement of levels and patterns of fluorescence indicative of complex formation is accomplished by fluorescence microscopy, preferably confocal fluorescence microscopy.
In a differential hybridization experiment, polynucleotide probes from two or more different biological samples are labeled with two or more different fluorescent labels with different emission wavelengths. Fluorescent signals are detected separately with different photomultipliers set to detect specific wavelengths. The relative abundances/expression levels of the polynucleotide probes in two or more samples is obtained.
Typically, microarray fluorescence intensities can be normalized to take into account variations in hybridization intensities when more than one microarray is used under similar test conditions. In a preferred embodiment, individual polynucleotide probe/target complex hybridization intensities are normalized using the intensities derived from internal normalization controls contained on each microarray.
The composition comprising a plurality of polynucleotide target sequences can be used as hybridizable elements in a microarray. Such a microarray can be employed to identify expression profiles associated with particular toxicological responses. Then, a particular subset of these polynucleotide targets can be identified whose expression is altered in response to a particular toxicological agent. These polynucleotides can be employed to identify other compounds with a similar toxicological response.
Alternatively, for some treatments with known side effects, the microarray, and expression patterns derived therefrom, is employed to xe2x80x9cfine tunexe2x80x9d the treatment regimen. A dosage is established that minimizes expression patterns associated with undesirable side effects. This approach may be more sensitive and rapid than waiting for the patient to show toxicological side effects before altering the course of treatment. Generally, the method for screening a compound or therapeutic treatment to identify those with a potential toxicological effect entails selecting a plurality of polynucleotide targets which consist of genes whose gene expression levels are altered in tissues treated with known toxicological agents when compared with untreated tissues and treating a sample with the compound to induce a pattern of gene expression comprising the expression of a plurality of polynucleotide probes. A test compound may be screened at several doses to determine doses which may be toxic and other doses that may not.
Then, the expression levels of the polynucleotide targets and the polynucleotide probes are compared to identify those compounds that induce expression levels of the polynucleotide probes that are similar to those of the polynucleotide targets. In one preferred embodiment, gene expression levels are compared by contacting the polynucleotide targets with the polynucleotide probes under conditions effective to form hybridization complexes between polynucleotide targets and polynucleotide probes; and detecting the presence or absence of the hybridization complexes.
Similarity may mean that at least 1, preferably at least 5, more preferably 10, of the upregulated polynucleotide targets form hybridization complexes with the polynucleotide probes at least once during a time course to a greater extent than would the probes of a sample not treated with the test compound. Similarity may also mean that at least 1, preferably at least 3, of the downregulated polynucleotide target sequences form hybridization complexes with the polynucleotide probes at least once during a time course to a lesser extent than would the probes of a sample not treated with the test compound.
Such a similarity of expression patterns means that a toxicological effect is associated with the compound or therapeutic treatment tested. Preferably, the toxicological agents belong to the class of polycyclic aromatic hydrocarbons, including benzo(a)pyrene, methylcholanthrene, benz(a)anthracene, 7,12-dimethylbenz(a)anthracene, their corresponding toxic metabolites and the like. Of particular interest is the study of the toxic effects of these test compounds on the liver, kidney, brain, spleen, pancreas and lung.